Method for Producing a Dairy Product

ABSTRACT

The present invention relates to a method for producing a dairy product using an enzyme having lactase activity.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/380,220 filed on Apr. 10, 2019, now pending, which is a continuationof U.S. application Ser. No. 16/021,048 filed on Jun. 28, 2018, now U.S.patent Ser. No. 10/306,902, which is a continuation of U.S. Ser. No.15/433,642 filed Feb. 15, 2017, now U.S. patent Ser. No. 10/058,107,which is a continuation of U.S. application Ser. No. 12/744,508 filedJul. 7, 2010, now abandoned, which is a 35 U.S.C. 371 nationalapplication of PCT/EP2008/066624 filed Dec. 2, 2008, which claimspriority or the benefit under 35 U.S.C. 119 of European application nos.07122110.5 and 08156674.7 filed Dec. 3, 2007 and May 21, 2008,respectively, and U.S. provisional application Nos. 61/055,164 and60/992,783 filed May 22, 2008 and Dec. 6, 2007, respectively. Thecontent of each application is fully incorporated herein by reference.

SEQUENCE LISTING

The present invention comprises a sequence listing, which isincorporated herein by reference.

TECHNICAL FIELD

The present invention relates to a method for producing a dairy productusing an enzyme having lactase activity.

BACKGROUND OF THE INVENTION

Lactose intolerance is perhaps the best-known food sensitivity in theUnited States and other parts of the world. It is estimated that about70% of the world's population has a genetically controlled limitedability to digest lactose. Therefore, to help dairy maldigesters keepdairy foods in their diet, there is a growing demand for dairy foodproducts that contain no or only low levels of lactose.

Lactase is used commercially to break down lactose in milk to producedairy products which are suitable for people with lactose intoleranceand/or have a sweeter taste. Because glucose and galactose are sweeterthan lactose, lactase produces a more pleasant taste. Lactase is alsoused in the manufacture of ice cream. Lactose crystallises at the lowtemperatures of ice cream, whereas glucose and galactose stay liquid andcontribute to a smoother texture. Lactase is also used in the conversionof whey into syrup. Lactase is also used for production of condensedmilk.

Lactases have been isolated from a large variety of organisms, includingmicroorganisms. Lactase is often an intracellular component ofmicroorganisms like Kluyveromyces and Bacillus. Kluyveromyces,especially K. fragilis and K. lactis, and other fungi such as those ofthe genera Candida, Torula and Torulopsis, are a common source of fungallactases, whereas B. coagulans and B. circulans are well known sourcesfor bacterial lactases. Several commercial lactase preparations derivedfrom these organisms are available such as Lactozym® (available fromNovozymes, Denmark), HA-Lactase (available from Chr. Hansen, Denmark)and Maxilact® (available from DSM, the Netherlands), all from K. lactis.All these lactases are so called neutral lactases having a pH optimumbetween pH 6 and pH 8. When such lactases are used in the production of,e.g., low-lactose yoghurt, the enzyme treatment will have to be done ina separate step before fermentation or rather high enzyme dosages haveto be used, because their activity drops as the pH decreases duringfermentation. Also, these lactases are not suitable for hydrolysis oflactose in milk performed at high temperature, which would in some casesbe beneficial to keep the microbial count low and thus ensure good milkquality.

Several extracellular lactases have been described having a lower pHoptimum, see, e.g., U.S. Pat. No. 5,736,374 which describes an exampleof such lactase, produced by Aspergillus oryzae.

A lactase from Bifidobacterium bifidum has been described having a hightransgalactosylating activity, both in the full-length form andespecially when truncated from the C-terminal end (see, e.g., Jørgensenet al., 2001, Appl. Microbiol. Biotechnol. 57: 647-652 or EP Patent No.1,283,876).

It is an object of the present invention to provide a method forproduction of dairy products, e.g., fermented dairy products, such asyoghurt, having a low level of lactose by using a lactase. It is also anobject to provide a method for production of low-lactose beverage milkhaving an extended shelf-life by using a lactase, where the method givesrise to low formation of off-flavour and/or low formation of brown coloras compared to known methods. Lactase to be used according to theinvention should hydrolyze lactose efficiently and optimally allow foralmost complete lactose hydrolysis. Especially, such lactase should havea high ratio of lactase to transgalactosylase activity. For use in theproduction of fermented dairy products, the lactase should be activeover a broad pH range.

SUMMARY OF THE INVENTION

The present inventors have surprisingly found that a C-terminallytruncated fragment of the extracellular lactase from Bifidobacteriumbifidum, which was originally isolated and patented for its ability tomake high amounts of galactooligosaccharides from lactose, can be usedvery successfully for hydrolysis of lactose in milk. When tested inwater+100 g/l lactose at 37° C., the enzyme makesgalactooligosaccharides with high efficiency as described in the priorart. However, when tested in milk, the ratio of hydrolytic totransgalactosylating activity has changed markedly, resulting inefficient hydrolysis and very low production of galactooligosaccharides.

Consequently, the present invention relates to a method for producing adairy product comprising

a) providing a milk-based substrate comprising lactose; and

b) treating said substrate with an enzyme having lactase activity andhaving an amino acid sequence which is at least 70% identical to aminoacids 28-1931 of SEQ ID NO: 1 or a fragment thereof.

In a preferred aspect, the invention relates to a method for producing adairy product comprising

a) providing a milk-based substrate comprising lactose; and

b) treating said substrate with an enzyme having lactase activity andhaving an amino acid sequence which is at least 70% identical to aminoacids 28-1331 of SEQ ID NO: 2.

Further, the inventors have surprisingly found that very low levels oflactose can be achieved when using the lactase from Bifidobacteriumbifidum as compared to other lactases typically used for treatment ofmilk. Another unexpected advantage of using the lactase fromBifidobacterium bifidum is that the enzyme is active at hightemperatures, allowing for treatment of the milk at, e.g., 52° C., thusreducing the microbial count and thereby improving the quality of themilk.

Therefore, in another aspect, the present invention relates to a methodfor producing a dairy product comprising

a) providing a milk-based substrate comprising lactose, and

b) treating said substrate with an enzyme having lactase activity,

where step b) takes place at a temperature of at least 50° C.

In a preferred embodiment, step b) takes place at a temperature of atleast 52° C.

Also, the inventors have surprisingly found that the lactase fromBifidobacterium bifidum is active over a broad pH range.

Therefore, in another aspect, the present invention relates to a methodfor producing a dairy product comprising

a) providing a milk-based substrate comprising lactose, and

b) treating said substrate with an enzyme having lactase activity, wherethe pH optimum of the lactase activity at 37° C. is above pH 5, andwhere the lactase activity of the enzyme at pH 5 is at least 50% of itslactase activity at pH 6 when measured at 37° C.

Use of a lactase enzyme being active over a broad pH spectrum isespecially useful for the production of fermented dairy products, whereit allows for low enzyme dosage, since the enzyme is still active duringand after fermentation. Also, very low levels of lactose in thefermented dairy product can be reached using such enzyme.

Therefore, in a preferred aspect, the present invention relates to amethod for producing a low-lactose fermented dairy product comprising

a) providing a milk-based substrate comprising lactose,

b) treating said substrate with an enzyme having lactase activity, wherethe pH optimum of the lactase activity at 37° C. is above pH 5, andwhere the lactase activity of the enzyme at pH 5 is at least 50% of itslactase activity at pH 6 when measured at 37° C., and

c) fermenting said substrate with a microorganism.

The present inventors have also surprisingly found that in themanufacture of low-lactose beverage milk having an extended shelf life,the lactose hydrolysis can preferentially be carried out at hightemperature, such as at a temperature of at least 60° C. Preferentially,such manufacture may comprise simultaneous low-pasteurization andlactase treatment. Therefore, in a preferred aspect, the presentinvention relates to a method for producing a dairy product comprising

a) providing a milk-based substrate comprising lactose, and

b) treating said substrate with an enzyme having lactase activity,

wherein step b) is performed for between 10 minutes and 4 hours at atemperature of between 62° C. and 64° C.

In a more preferred aspect, step b) in such method is followed bycooling to below 10° C. without further heat treatment. This will allowfor the enzyme to be still active after the milk has been cooled, i.e.,during its storage. In another more preferred aspect, step b) in suchmethod is followed by UHT treatment.

Preferably, in the methods of the invention, at least 70% of the lactosein the milk-based substrate is hydrolyzed. More preferably, at least80%, such as at least 85%, at least 90%, at least 95% or at least 98%,of the lactose in the milk-based substrate is hydrolyzed.

In another aspect, the present invention relates to a method forproducing a dairy product comprising

a) providing a milk-based substrate comprising lactose; and

b) treating said substrate with an enzyme having lactase activity andhaving an amino acid sequence which is at least 70% identical to SEQ IDNO: 3 or a fragment thereof.

In another aspect, the present invention relates to a method forproducing a dairy product comprising

a) providing a milk-based substrate comprising lactose; and

b) treating said substrate with an enzyme having lactase activity andhaving an amino acid sequence which is at least 70% identical to SEQ IDNO: 4 or a fragment thereof.

In another aspect, the present invention relates to a method forproducing a dairy product comprising

a) providing a milk-based substrate comprising lactose; and

b) treating said substrate with an enzyme having lactase activity andhaving an amino acid sequence which is at least 70% identical to SEQ IDNO: 5 or a fragment thereof.

In another aspect, the present invention relates to a method forproducing a dairy product comprising

a) providing a milk-based substrate comprising lactose; and

b) treating said substrate with an enzyme having lactase activity andhaving an amino acid sequence which is at least 70% identical to SEQ IDNO: 6 or a fragment thereof.

In yet another aspect, the present invention relates to a method forproducing a dairy product comprising

a) providing a milk-based substrate comprising lactose; and

b) treating said substrate with an enzyme having lactase activity andhaving an amino acid sequence which is at least 70% identical to SEQ IDNO: 7 or a fragment thereof.

DETAILED DISCLOSURE OF THE INVENTION Milk-Based Substrate

The term “milk”, in the context of the present invention, is to beunderstood as the lacteal secretion obtained by milking any mammal, suchas cows, sheep, goats, buffaloes or camels.

“Milk-based substrate”, in the context of the present invention, may beany raw and/or processed milk material. Useful milk-based substratesinclude, but are not limited to solutions/suspensions of any milk ormilk like products comprising lactose, such as whole or low fat milk,skim milk, buttermilk, reconstituted milk powder, condensed milk,solutions of dried milk, UHT milk, whey, whey permeate, acid whey, orcream.

Preferably, the milk-based substrate is milk or an aqueous solution ofskim milk powder.

The milk-based substrate may be more concentrated than raw milk.

In one embodiment, the milk-based substrate has a ratio of protein tolactose of at least 0.2, preferably at least 0.3, at least 0.4, at least0.5, at least 0.6 or, most preferably, at least 0.7.

The milk-based substrate may be homogenized and pasteurized according tomethods known in the art.

“Homogenizing” as used herein means intensive mixing to obtain a solublesuspension or emulsion. It may be performed so as to break up the milkfat into smaller sizes so that it no longer separates from the milk.This may be accomplished by forcing the milk at high pressure throughsmall orifices.

“Pasteurizing” as used herein means reducing or eliminating the presenceof live organisms, such as microorganisms, in the milk-based substrate.Preferably, pasteurization is attained by maintaining a specifiedtemperature for a specified period of time. The specified temperature isusually attained by heating. The temperature and duration may beselected in order to kill or inactivate certain bacteria, such asharmful bacteria, and/or to inactivate enzymes in the milk. A rapidcooling step may follow.

Dairy Product

A “dairy product” in the context of the present invention may be anyfood product wherein one of the major constituents is milk-based.Preferable, the major constituent is milk-based. More preferably, themajor constituent is a milk-based substrate which has been treated withan enzyme having lactase activity according to a method of theinvention. In the context of the present invention “one of the majorconstituents” means a constituent having a dry matter which constitutesmore than 20%, preferably more than 30% or more than 40% of the totaldry matter of the dairy product, whereas “the major constituent” means aconstituent having a dry matter which constitutes more than 50%,preferably more than 60% or more than 70% of the total dry matter of thedairy product.

A dairy product according to the invention may be, e.g., skim milk, lowfat milk, whole milk, cream, UHT milk, milk having an extended shelflife, a fermented milk product, cheese, yoghurt, butter, dairy spread,butter milk, acidified milk drink, sour cream, whey based drink, icecream, condensed milk, dulce de leche or a flavoured milk drink. A dairyproduct may be manufactured by any method known in the art.

A dairy product may additionally comprise non-milk components, e.g.,vegetable components such as, e.g., vegetable oil, vegetable protein,and/or vegetable carbohydrates. Dairy products may also comprise furtheradditives such as, e.g., enzymes, flavouring agents, microbial culturessuch as probiotic cultures, salts, sweeteners, sugars, acids, fruit,fruit juices, or any other component known in the art as a component of,or additive to, a dairy product.

In one embodiment of the invention, one or more milk components and/ormilk fractions ac-count for at least 50% (weight/weight), such as atleast 70%, e.g., at least 80%, preferably at least 90%, of the dairyproduct.

In one embodiment of the invention, one or more milk-based substrateshaving been treated with an enzyme having lactase activity according toa method of the invention account for at least 50% (weight/weight), suchas at least 70%, e.g., at least 80%, preferably at least 90%, of thedairy product.

In one embodiment of the invention, the dairy product is a dairy productwhich is not enriched by addition of galactooligosaccharides.

In one embodiment of the invention, the enzyme-treated milk-basedsubstrate is not dried before being used as an ingredient in the dairyproduct.

In one embodiment of the invention, the dairy product is ice cream. Inthe present context, ice cream may be any kind of ice cream such as fullfat ice cream, low fat ice cream, or ice cream based on yoghurt or otherfermented milk products. Ice cream may be manufactured by any methodknown in the art.

In one embodiment of the invention, the dairy product is milk orcondensed milk.

In one preferred embodiment of the invention, the dairy product is UHTmilk. UHT milk in the context of the present invention is milk which hasbeen subjected to a sterilization procedure which is intended to killall microorganisms, including the bacterial spores. UHT (ultra hightemperature) treatment may be, e.g., heat treatment for 30 seconds at130° C., or heat treatment for one second at 145° C.

In one preferred embodiment of the invention, the dairy product is ESLmilk. ESL milk in the context of the present invention is milk which hasan extended shelf life due to microfiltration and/or heat treatment andwhich is able to stay fresh for at least 15 days, preferably for atleast 20 days, on the store shelf at 2-5° C.

In another preferred embodiment of the invention, the dairy product is afermented dairy product, e.g., yoghurt.

Fermented Dairy Product

A “fermented dairy product” in the context of the present invention isto be understood as any dairy product wherein any type of fermentationforms part of the production process. Examples of fermented dairyproducts are products like yoghurt, buttermilk, creme fraiche, quark andfromage frais. A fermented dairy product may be produced by any methodknown in the art.

“Fermentation” in the method of the present invention means theconversion of carbohydrates into alcohols or acids through the action ofa microorganism. Preferably, fermentation in the method of the presentinvention comprises conversion of lactose to lactic acid.

In the context of the present invention, “microorganism” may include anybacterium or fungus being able to ferment the milk substrate.

The microorganisms used for most fermented milk products are selectedfrom the group of bacteria generally referred to as lactic acidbacteria. As used herein, the term “lactic acid bacterium” designates agram-positive, microaerophilic or anaerobic bacterium, which fermentssugars with the production of acids including lactic acid as thepredominantly produced acid, acetic acid and propionic acid. Theindustrially most useful lactic acid bacteria are found within the order“Lactobacillales” which includes Lactococcus spp., Streptococcus spp.,Lactobacillus spp., Leuconostoc spp., Pseudoleuconostoc spp.,Pediococcus spp., Brevibacterium spp., Enterococcus spp. andPropionibacterium spp. Additionally, lactic acid producing bacteriabelonging to the group of anaerobic bacteria, bifidobacteria, i.e.,Bifidobacterium spp., which are frequently used as food cultures aloneor in combination with lactic acid bacteria, are generally included inthe group of lactic acid bacteria.

Lactic acid bacteria are normally supplied to the dairy industry eitheras frozen or freeze-dried cultures for bulk starter propagation or asso-called “Direct Vat Set” (DVS) cultures, intended for directinoculation into a fermentation vessel or vat for the production of afermented dairy product. Such cultures are in general referred to as“starter cultures” or “starters”.

Commonly used starter culture strains of lactic acid bacteria aregenerally divided into mesophilic organisms having optimum growthtemperatures at about 30° C. and thermophilic organisms having optimumgrowth temperatures in the range of about 40 to about 45° C. Typicalorganisms belonging to the mesophilic group include Lactococcus lactis,Lactococcus lactis subsp. cremoris, Leuconostoc mesenteroides subsp.cremoris, Pseudoleuconostoc mesenteroides subsp. cremoris, Pediococcuspentosaceus, Lactococcus lactis subsp. lactis biovar. diacetylactis,Lactobacillus casei subsp. casei and Lactobacillus paracasei subsp.paracasei. Thermophilic lactic acid bacterial species include asexamples Streptococcus thermophilus, Enterococcus faecium, Lactobacillusdelbrueckii subsp. lactis, Lactobacillus helveticus, Lactobacillusdelbrueckii subsp. bulgaricus and Lactobacillus acidophilus.

Also the anaerobic bacteria belonging to the genus Bifidobacteriumincluding Bifidobacterium bifidum, Bifidobacterium animalis andBifidobacterium Iongum are commonly used as dairy starter cultures andare generally included in the group of lactic acid bacteria.Additionally, species of Propionibacteria are used as dairy startercultures, in particular, in the manufacture of cheese. Additionally,organisms belonging to the Brevibacterium genus are commonly used asfood starter cultures.

Another group of microbial starter cultures are fungal cultures,including yeast cultures and cultures of filamentous fungi, which areparticularly used in the manufacture of certain types of cheese andbeverage. Examples of fungi include Penicillium roqueforti, Penicilliumcandidum, Geotrichum candidum, Torula kefir, Saccharomyces kefir andSaccharomyces cerevisiae.

In one embodiment of the present invention, the microorganism used forfermentation of the milk-based substrate is Lactobacillus casei or amixture of Streptococcus thermophilus and Lactobacillus delbrueckiisubsp. bulgaricus.

Fermentation processes to be used in a method of the present inventionare well known and the person of skill in the art will know how toselect suitable process conditions, such as temperature, oxygen, amountand characteristics of microorganism/s, additives such as, e.g.,carbohydrates, flavours, minerals, enzymes, and process time. Obviously,fermentation conditions are selected so as to support the achievement ofthe present invention.

As a result of fermentation, pH of the milk-based substrate will belowered. The pH of a fermented dairy product of the invention may be,e.g., in the range 3.5-6, such as in the range 3.5-5, preferably in therange 3.8-4.8.

In a preferred embodiment, the fermented dairy product is yoghurt.

Method for Producing a Dairy Product

As mentioned above, the present invention in one aspect relates to amethod for producing a dairy product comprising:

a) providing a milk-based substrate comprising lactose; and

b) treating said substrate with an enzyme having lactase activity andhaving an amino acid sequence which is at least 70% identical to aminoacids 28-1931 of SEQ ID NO: 1 or a fragment thereof.

In a preferred aspect, the invention relates to a method for producing adairy product comprising

a) providing a milk-based substrate comprising lactose; and

b) treating said substrate with an enzyme having lactase activity andhaving an amino acid sequence which is at least 70% identical to aminoacids 28-1331 of SEQ ID NO: 2.

In another aspect, the invention relates to a method for producing adairy product comprising

a) providing a milk-based substrate comprising lactose; and

b) treating said substrate with an enzyme having lactase activity andhaving an amino acid sequence which is at least 70% identical to SEQ IDNO: 3 or a fragment thereof.

In another aspect, the invention relates to a method for producing adairy product comprising

a) providing a milk-based substrate comprising lactose; and

b) treating said substrate with an enzyme having lactase activity andhaving an amino acid sequence which is at least 70% identical to SEQ IDNO: 4 or a fragment thereof.

In another aspect, the invention relates to a method for producing adairy product comprising

a) providing a milk-based substrate comprising lactose; and

b) treating said substrate with an enzyme having lactase activity andhaving an amino acid sequence which is at least 70% identical to SEQ IDNO: 5 or a fragment thereof.

In another aspect, the invention relates to a method for producing adairy product comprising

a) providing a milk-based substrate comprising lactose; and

b) treating said substrate with an enzyme having lactase activity andhaving an amino acid sequence which is at least 70% identical to SEQ IDNO: 6 or a fragment thereof.

In another aspect, the invention relates to a method for producing adairy product comprising

a) providing a milk-based substrate comprising lactose; and

b) treating said substrate with an enzyme having lactase activity andhaving an amino acid sequence which is at least 70% identical to SEQ IDNO: 7 or a fragment thereof.

In another aspect, the invention relates to a method for producing adairy product comprising

a) providing a milk-based substrate comprising lactose, and

b) treating said substrate with an enzyme having lactase activity,wherein step b) takes place at a temperature of at least 50° C.

In yet another aspect, the invention relates to a method for producing adairy product comprising

a) providing a milk-based substrate comprising lactose,

b) treating said substrate with an enzyme having lactase activity, wherethe pH optimum of the lactase activity at 37° C. is above pH 5, andwhere the lactase activity of the enzyme at pH 5 is at least 50% of itslactase activity at pH 6 when measured at 37° C.

The skilled person will know how to determine the lactase activity atdifferent pH and thereby determine the pH optimum for the enzyme. Thelactase activity at different pH may be determined by measuringhydrolysis of lactose at 37° C. for 30 minutes, preferably in a buffercomprising succinate, HEPES, CHES, KCl, CaCl₂ and MgCl₂, e.g., by usinga method as described in the Examples of the present application. Forthe avoidance of doubt, HEPES is a buffering agent,4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, and CHES is abuffering agent, N-Cyclohexyl-2-aminoethanesulfonic acid.

The enzyme-treated milk-based substrate may optionally be mixed withother ingredients to obtain the dairy product. In one embodiment of theinvention, the enzyme-treated milk-based substrate is mixed with otheringredients to obtain the dairy product.

In one embodiment of the invention, the dairy product is milk. Inanother embodiment, the dairy product is condensed milk. In anotherembodiment, the dairy product is ice cream. In another embodiment, thedairy product is UHT milk. In another embodiment, the dairy product isESL milk. In yet another embodiment, the dairy product is a fermenteddairy product, e.g., yoghurt.

Preferably, the dairy product is a low-lactose dairy product.“Low-lactose”, in the context of the present invention, means that theamount of lactose in the dairy product, such as in the fermented dairyproduct, has been reduced by at least 70%, preferably 80%, 90%, 95%,98%, 99% or 99,5%.

Method for Producing a Low-Lactose Fermented Dairy Product

One embodiment of the present invention relates to a method forproducing a low-lactose fermented dairy product comprising

a) providing a milk-based substrate comprising lactose,

b) treating said substrate with an enzyme having lactase activity, wherethe lactase activity of the enzyme at pH 5 is at least 50% of itslactase activity at pH 6 when measured at 37° C., and

c) fermenting said substrate with a microorganism.

Another embodiment of the invention relates to a method for producing alow-lactose fermented dairy product comprising

a) providing a milk-based substrate comprising lactose,

b) treating said substrate with an enzyme having lactase activity andhaving an amino acid sequence which is at least 70% identical to SEQ IDNO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ IDNO: 6, SEQ ID NO: 7, or a fragment of any of these, and

c) fermenting said substrate with a microorganism.

Preferably, in these embodiments, step b) and step c) are performedessentially at the same time.

In the context of the method of the invention, “essentially at the sametime” means that enzyme treatment and fermentation are not performed asseparate steps, i.e., incubation of the milk-based substrate with theenzyme is not performed as a separate step before inoculation with themicroorganism. Instead, the enzyme and the microorganism may be added tothe milk-based substrate at essentially the same time. I.e., the enzymemay be added to the milk-based substrate immediately before inoculationwith the microorganism. “Immediately before” in this context meanswithout a separate incubation step for the enzymatic hydrolysis.Alternatively, the microorganism may be added immediately before theenzyme, or the microorganism and the enzyme may be added at the sametime. “Essentially at the same time” in the context of the method of theinvention may mean that the enzyme is active throughout the whole ofstep c).

“Essentially at the same time” does not mean that the enzymatichydrolysis of the lactose in the milk-based substrate is completed whenthe fermentation is completed, i.e., when the pH has dropped to a levelpreventing further fermenting activity of the microbial starter culture.

In a preferred embodiment of the invention, the enzyme is still activeafter completion of step c). In a more preferred embodiment, the enzymehas retained at least 20%, such as at least 30%, at least 40%, at least50%, at least 60% or at least 70%, of its lactase activity aftercompletion of step c) as compared to its activity when added to themilk-based substrate.

After completion of step c) may mean when at least one of the below istrue:

temperature is lowered to below 30° C.

pH has reached 4.55

pH does no longer decrease by more than 0.2 units per hour.

In another preferred embodiment, less than 80%, such as less than 70%,less than 60%, less than 50%, less than 40%, less than 30% or less than20%, of the lactose has been hydrolyzed after two hours of fermentation.“After two hours of fermentation” in the context of the invention meansthat the milk-based substrate, after having been inoculated with themicroorganism, has been incubated for two hours at a temperature whichis appropriate for the fermentation process.

In another preferred embodiment, less than 80%, such as less than 70%,less than 60%, less than 50%, less than 40%, less than 30% or less than20%, of the lactose has been hydrolyzed when pH of the milk-basedsubstrate has dropped to pH 5.

In yet another preferred embodiment, less than 80%, such as less than70%, less than 60%, less than 50%, less than 40%, less than 30% or lessthan 20%, of the lactose has been hydrolyzed when step c) is completed.

In a preferred embodiment, more than 70%, such as more than 80%, morethan 90%, more than 95%, more than 97%, more than 98% or more than 99%,of the lactose in the milk-based substrate has been hydrolyzed two days,i.e., 48 hours, after start of the fermentation. “Start of thefermentation” is when the milk-based substrate has been inoculated withthe microorganism and incubated at a temperature which is appropriatefor the fermentation process.

In another preferred embodiment, more than 70%, such as more than 80%,more than 90%, more than 95%, more than 97%, more than 98% or more than99%, of the lactose in the milk-based substrate has been hydrolyzed twodays, i.e., 48 hours, after completion of step c).

In another preferred embodiment, more than 70%, such as more than 80%,more than 90%, more than 95%, more than 97%, more than 98% or more than99%, of the lactose in the milk-based substrate has been hydrolyzed inthe final fermented dairy product. The “final fermented dairy product”is the fermented dairy product as sold to the consumer of the product.

In the embodiments of the invention where the milk-based substrate isbeing fermented, the enzyme treatment is preferably conducted at thenatural pH of the milk-based substrate during the fermentation process,i.e., the pH will gradually decrease from the natural pH of theunfermented milk-based substrate to the pH of the fermented milk-basedsubstrate. In such aspect, the enzyme treatment is preferably conductedat an appropriate temperature for the fermentation process.

Method for Producing a Low-Lactose Beverage Milk Product Having anExtended Shelf Life

One embodiment of the present invention relates to a method forproducing a low-lactose milk product comprising

a) providing a milk-based substrate comprising lactose, and

b) treating said substrate with an enzyme having lactase activity,

wherein step b) takes place at a temperature of at least 60° C.

In a preferred embodiment, step b) takes place at a temperature of atleast 62° C., such as at least 63° C., more preferred at a temperatureof at least 64° C., such as at least 65° C., at least 67° C. or at least70° C., and most preferred at a temperature of at least 75° C.

The low-lactose milk product may be beverage milk having a longer shelflife than fresh milk which normally has a shelf life of 4-7 days. It mayhave an improved quality as compared to other low-lactose beverage milkproducts having a longer shelf life. It may, e.g., have a lowermicrobial count, a less bitter taste and/or a less brown colour.

Preferably, the milk product is ESL milk. More preferably, the milkproduct is UHT milk.

In a preferred aspect, the milk-based substrate is raw milk. In anotherpreferred aspect, the milk-based substrate, preferably raw milk, has notbeen pasteurized before step b).

In a preferred aspect, no pasteurization of the enzyme treatedmilk-based substrate is performed after step b).

In a preferred aspect, microfiltration of the milk-based substrate isperformed before step b). In that case, the enzyme should preferentiallybe sterile. In another preferred aspect, microfiltration of the enzymetreated milk-based substrate is performed after step b).

In a preferred aspect, step b) is performed for between 10 minutes and 4hours at a temperature of between 62° C. and 64° C. In a more preferredaspect, step b) is performed for between 20 minutes and 2 hours at atemperature of between 62° C. and 64° C. In an even more preferredaspect, step b) is performed for between 20 and 60 minutes, such as forabout 30 minutes, at a temperature of about 63° C. Such simultaneous lowpasteurization and lactase treatment of, e.g., raw milk will give riseto low-lactose beverage milk having a higher quality as compared tolow-lactose beverage milk where lactase treatment has been performed atlow temperature, e.g., at 5° C. for up to 24 hours as is often used inthe dairy industry.

In a more preferred aspect, step b) is followed by cooling to below 10°C. without further heat treatment. This will allow for the enzyme to bestill active after the milk has been cooled, i.e., during its storage.Preferably, less than 80% of the lactose has been hydrolyzed when stepb) is completed, and more than 90% of the lactose has been hydrolyzedafter one week. More preferably, less than 60% of the lactose has beenhydrolyzed when step b) is completed, and more than 95% of the lactosehas been hydrolyzed after one week

In another more preferred aspect, step b) is followed by UHT treatment.

Enzyme Having Lactase Activity

In step b) in the methods of the present invention, the milk-basedsubstrate is treated with an enzyme having lactase activity.

A lactase in the context of the present invention is any glycosidehydrolase having the ability to hydrolyse the disaccharide lactose intoconstituent galactose and glucose monomers. The group of lactasescomprises but is not limited to enzymes assigned to subclass EC3.2.1.108. Enzymes assigned to other subclasses, such as, e.g., EC3.2.1.23, may also be lactases in the context of the present invention.A lactase in the context of the invention may have other activities thanthe lactose hydrolyzing activity, such as for example atransgalactosylating activity. In the context of the invention, thelactose hydrolyzing activity of the lactase may be referred to as itslactase activity or its beta-galactosidase activity.

Enzymes having lactase activity to be used in a method of the presentinvention may be of animal, of plant or of microbial origin. Preferredenzymes are obtained from microbial sources, in particular from afilamentous fungus or yeast, or from a bacterium.

The enzyme may, e.g., be derived from a strain of Agaricus, e.g., A.bisporus; Ascovaginospora; Aspergillus, e.g., A. niger, A. awamori, A.foetidus, A. japonicus, A. oryzae; Candida; Chaetomium; Chaetotomastia;Dictyostelium, e.g., D. discoideum; Kluveromyces, e.g., K. fragilis, K.lactis; Mucor, e.g., M. javanicus, M. mucedo, M. subtilissimus;Neurospora, e.g., N. crassa; Rhizomucor, e.g., R. pusillus; Rhizopus,e.g., R. arrhizus, R. japonicus, R. stolonifer; Scierotinia, e.g., S.libertiana; Torula; Torulopsis; Trichophyton, e.g., T. rubrum;Whetzelinia, e.g., W. sclerotiorum; Bacillus, e.g., B. coagulans, B.circulans, B. megaterium, B. novalis, B. subtilis, B. pumilus, B.stearothermophilus, B. thuringiensis; Bifidobacterium, e.g., B. longum,B. bifidum, B. animalis; Chryseobacterium; Citrobacter, e.g., C.freundii; Clostridium, e.g,. C. perfringens; Diplodia, e.g., D.gossypina; Enterobacter, e.g., E. aerogenes, E. cloacae Edwardsiella, E.tarda; Erwinia, e.g., E. herbicola; Escherichia, e.g., E. coli;Klebsiella, e.g., K. pneumoniae; Miriococcum; Myrothesium; Mucor;Neurospora, e.g., N. crassa; Proteus, e.g., P. vulgaris; Providencia,e.g., P. stuartii; Pycnoporus, e.g., Pycnoporus cinnabarinus, Pycnoporussanguineus; Ruminococcus, e.g., R. torques; Salmonella, e.g., S.typhimurium; Serratia, e.g., S. liquefasciens, S. marcescens; Shigella,e.g., S. flexneri; Streptomyces, e.g., S. antibioticus, S.castaneoglobisporus, S. violeceoruber; Trametes; Trichoderma, e.g., T.re,esei, T. viride; Yersinia, e.g., Y. enterocolitica.

In a preferred embodiment, the enzyme is a lactase from a bacterium,e.g., from the family Bifidobacteriaceae, such as from the genusBifidobacterium, such as from a strain of B. bifidum, B. animalis or B.longum. In a more preferred embodiment, the enzyme is a lactase fromBifidobacterium bifidum. A preferred enzyme is a lactase having asequence which is at least 50%, such as at least 60%, at least 70%, atleast 80%, at least 90%, at least 95% or at least 98% identical to aminoacids 28-1931 of SEQ ID NO: 1 or to a lactase active fragment thereof.Such lactase active fragment of SEQ ID NO: 1 may be any fragment of SEQID NO: 1 having lactase activity. A lactase active fragment of SEQ IDNO: 1 may be, e.g., amino acids 28-979, amino acids 28-1170, amino acids28-1323, amino acids 28-1331, or amino acids 28-1600 of SEQ ID NO: 1.

In a preferred embodiment, an enzyme having lactase activity to be usedin a method of the present invention comprises an amino acid sequencewhich is at least 50% identical to amino acids 28-1331 of SEQ ID NO: 2.In a more preferred embodiment, the enzyme comprises an amino acidsequence which is at least 60%, such as at least 70%, at least 80%, atleast 90%, at least 95% or at least 98% identical to amino acids 28-1331of SEQ ID NO: 2.

In another preferred embodiment, an enzyme having lactase activity to beused in a method of the present invention has an amino acid sequencewhich is at least 50% identical to amino acids 28-1331 of SEQ ID NO: 2.In a more preferred embodiment, the enzyme has an amino acid sequencewhich is at least 60%, such as at least 70%, at least 80%, at least 90%,at least 95% or at least 98% identical to amino acids 28-1331 of SEQ IDNO: 2.

In another embodiment, an enzyme having lactase activity to be used in amethod of the present invention has an amino acid sequence which is atleast 50% identical to SEQ ID NO: 3. Preferably, the enzyme has an aminoacid sequence which is at least 60%, such as at least 70%, at least 80%,at least 90%, at least 95% or at least 98% identical to SEQ ID NO: 3.

In another embodiment, an enzyme having lactase activity to be used in amethod of the present invention has an amino acid sequence which is atleast 50% identical to SEQ ID NO: 4. Preferably, the enzyme has an aminoacid sequence which is at least 60%, such as at least 70%, at least 80%,at least 90%, at least 95% or at least 98% identical to SEQ ID NO: 4.

In another embodiment, an enzyme having lactase activity to be used in amethod of the present invention has an amino acid sequence which is atleast 50% identical to SEQ ID NO: 5. Preferably, the enzyme has an aminoacid sequence which is at least 60%, such as at least 70%, at least 80%,at least 90%, at least 95% or at least 98% identical to SEQ ID NO: 5.

In another embodiment, an enzyme having lactase activity to be used in amethod of the present invention has an amino acid sequence which is atleast 50% identical to SEQ ID NO: 6. Preferably, the enzyme has an aminoacid sequence which is at least 60%, such as at least 70%, at least 80%,at least 90%, at least 95% or at least 98% identical to SEQ ID NO: 6.

In another embodiment, an enzyme having lactase activity to be used in amethod of the present invention has an amino acid sequence which is atleast 50% identical to SEQ ID NO: 7. Preferably, the enzyme has an aminoacid sequence which is at least 60%, such as at least 70%, at least 80%,at least 90%, at least 95% or at least 98% identical to SEQ ID NO: 7.

For purposes of the present invention, the degree of identity betweentwo amino acid sequences is determined using the Needleman-Wunschalgorithm (Needleman and Wunsch (1970) J. Mol. Biol. 48: 443-453) asimplemented in the Needle program of the EMBOSS package (EMBOSS: TheEuropean Molecular Biology Open Software Suite, Rice et al. (2000)Trends in Genetics 16: 276-277), preferably version 3.0.0 or later. Theoptional parameters used are gap open penalty of 10, gap extensionpenalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62)substitution matrix. The output of Needle labelled “longest identity”(obtained using the-no brief option) is used as the percent identity andis calculated as follows:

(Identical Residues×100)/(Length of Alignment−Total Number of Gaps inAlignment)

Lactases to be used in a method of the present invention may beextracellular. They may have a signal sequence at their N-terminus,which is cleaved off during secretion.

Lactases to be used in a method of the present invention may be derivedfrom any of the sources mentioned herein. The term “derived” means inthis context that the enzyme may have been isolated from an organismwhere it is present natively, i.e., the identity of the amino acidsequence of the enzyme are identical to a native enzyme. The term“derived” also means that the enzymes may have been producedrecombinantly in a host organism, the recombinantly produced enzymehaving either an identity identical to a native enzyme or having amodified amino acid sequence, e.g., having one or more amino acids whichare deleted, inserted and/or substituted, i.e., a recombinantly producedenzyme which is a mutant and/or a fragment of a native amino acidsequence. Within the meaning of a native enzyme are included naturalvariants. Furthermore, the term “derived” includes enzymes producedsynthetically by, e.g., peptide synthesis. The term “derived” alsoencompasses enzymes which have been modified, e.g., by glycosylation,phosphorylation etc., whether in vivo or in vitro. With respect torecombinantly produced enzyme the term “derived from” refers to theidentity of the enzyme and not the identity of the host organism inwhich it is produced recombinantly.

The lactase may be obtained from a microorganism by use of any suitabletechnique. For instance, a lactase enzyme preparation may be obtained byfermentation of a suitable microorganism and subsequent isolation of alactase preparation from the resulting fermented broth or microorganismby methods known in the art. The lactase may also be obtained by use ofrecombinant DNA techniques. Such method normally comprises cultivationof a host cell transformed with a recombinant DNA vector comprising aDNA sequence encoding the lactase in question and the DNA sequence beingoperationally linked with an appropriate expression signal such that itis capable of expressing the lactase in a culture medium underconditions permitting the expression of the enzyme and recovering theenzyme from the culture. The DNA sequence may also be incorporated intothe genome of the host cell. The DNA sequence may be of genomic, cDNA orsynthetic origin or any combinations of these, and may be isolated orsynthesized in accordance with methods known in the art.

Lactases to be used in a method of the present invention may bepurified. The term “purified” as used herein covers lactase enzymeprotein essentially free from insoluble components from the productionorganism. The term “purified” also covers lactase enzyme proteinessentially free from insoluble components from the native organism fromwhich it is obtained. Preferably, it is also separated from some of thesoluble components of the organism and culture medium from which it isderived. More preferably, it is separated by one or more of the unitoperations: filtration, precipitation, or chromatography.

Accordingly, the enzyme having lactase activity may be purified, viz.only minor amounts of other proteins being present. The expression“other proteins” relate in particular to other enzymes. The term“purified” as used herein also refers to removal of other components,particularly other proteins and most particularly other enzymes presentin the cell of origin of the lactase. The lactase may be “substantiallypure”, i.e., free from other components from the organism in which it isproduced, i.e., e.g., a host organism for recombinantly producedlactase. Preferably, the lactase is an at least 40% (w/w) pure enzymeprotein preparation, more preferably at least 50%, 60%, 70%, 80% or evenat least 90% pure.

The term enzyme having lactase activity includes whatever auxiliarycompounds that may be necessary for the enzyme's catalytic activity,such as, e.g., an appropriate acceptor or cofactor, which may or may notbe naturally present in the reaction system.

The enzyme may be in any form suited for the use in question, such as,e.g., in the form of a dry powder or granulate, a non-dusting granulate,a liquid, a stabilized liquid, or a protected enzyme.

The enzyme is added in a suitable amount to achieve the desired degreeof lactose hydrolysis under the chosen reaction conditions. The enzymemay be added at a concentration of between 100 and 5000 LAU per litremilk-based substrate, preferably less than 3000, such as less than 1500,less than 1000, less than 750 or less than 500, LAU per litre milk-basedsubstrate.

In a preferred embodiment, the enzyme is added at a concentration ofbetween 5 and 100 LAU per g lactose in the milk-based substrate,preferably less than 50, such as less than 40, less than 30, less than20 or less than 10, LAU per g lactose in the milk-based substrate.

In the context of the present application, 1 lactase unit (1 LAU) is theamount of enzyme which releases 1 micromole glucose per minute inM-buffer at pH 6.5 and 37° C. with a lactose concentration of 4.75% w/v.M-buffer is prepared by dissolving 3.98 g C₆H₅Na₃O₇-2H₂O, 8.31 g citricacid, 0.9 g K₂SO₄, 2.6 g K₂HPO₄, 7.35 g KH₂PO₄, 5.45 g KOH, 4.15 gMgCl₂-6H₂O, 3.75 g CaCl₂-2H₂O and 1.4 g NaHCO₃ in 4 litre water, adding12.5 ml 4 N NaOH, adjusting to pH 6.5 using HCl, and adding water up toa total volume of 5 liter.

The activity in LAU of a specific lactase may be determined by directmeasurement of glucose released from lactose under the conditionsdescribed above. The skilled person will know how to determine suchactivity. Alternatively, the activity may be determined by using thelactase activity assay described in Example 1 of the presentapplication. Here, the activity is obtained by comparing to a standardcurve run with a lactase of known activity, and the activity of theunknown sample calculated from this. The lactase of known activity may,e.g., be Lactozym obtained from Novozymes A/S, Denmark.

In a preferred embodiment, the enzyme having lactase activity to be usedin a method of the present invention has a lactase activity at 37° C.and pH 5 which is at least 55%, such as at least 60%, at least 65%, atleast 70% or at least 75%, of its lactase activity at 37° C. and pH 6.

In another preferred embodiment, the enzyme having lactase activity tobe used in a method of the present invention has a lactase activity at37° C. and pH 4.5 which is at least 10%, such as at least 20%, at least30%, at least 35% or at least 40%, of its lactase activity at 37° C. andpH 6.

In another preferred embodiment, the enzyme having lactase activity tobe used in a method of the present invention has a pH optimum of thelactase activity at 37° C. which is above pH 5.5.

In another preferred embodiment, the enzyme having lactase activity tobe used in a method of the present invention has a lactase activity at atemperature of 52° C. and a pH of 6.5 which is at least 50%, such as atleast 55%, at least 60%, at least 65%, at least 70%, at least 75% or atleast 80%, of its lactase activity at a temperature of 38° C. and a pHof 6.5.

The skilled person will know how to determine the lactase activity atdifferent pH and temperature. The lactase activity at different pH andtemperature is preferably determined by using a method as described inthe Examples of the present application.

In a preferred embodiment of the present invention, Km of the enzymehaving lactase activity at 5° C. is below 25 mM, such as below 20 mM,below 15 mM or below 10 mM. In another preferred embodiment, Km of theenzyme having lactase activity at 37° C. is below 25 mM, such as below20 mM or below 15 mM. The skilled person will know how to determine Kmfor the lactase activity at a specific temperature. Km may be determinedby the method used in the Examples of the present application.

In another preferred embodiment, the enzyme when hydrolyzing the lactosein the milk-based substrate has a ratio of lactase to transgalactosylaseactivity of more than 1:1, such as more than 2:1 or more than 3:1. Inanother preferred embodiment, the enzyme treatment is performed underconditions where the lactase activity of the enzyme is higher than thetransgalactosylase activity, such as at least two times higher or atleast three times higher.

The ratio of lactase to transgalactosylase activity in the milk-basedsubstrate may, e.g., be determined by HPLC analysis. In anotherpreferred embodiment, the enzyme treatment is performed under conditionswhere at least 50% (w/w %) of the hydrolyzed lactose is converted intofree galactose. In another preferred embodiment, the enzyme treatment isperformed under conditions where the hydrolyzed lactose is convertedinto equal amounts of free glucose and free galactose.

Example 1 Lactase Activity-Assay in Eppendorf Tubes at 37° C., pH 6.5Principle:

Lactase hydrolyzes lactose into glucose and galactose. Glucose ismeasured after a modified version of the common glucoseoxidase/peroxidase assay (Werner et al., 1970, Z. Analyt. Chem. 252:224).

LAU is defined as the amount of enzyme liberating 1 micromole of glucoseper min at 37° C., pH 6.5 in M-buffer (M-buffer is defined in thedescription of the present patent application). Alternatively, theactivity in LAU for a specific lactase may be determined by the methoddescribed here. The value obtained is compared to a standard curve runwith a lactase of known activity, and the activity of the unknown samplecalculated from this. The lactase of known activity may, e.g., beLactozym obtained from Novozymes A/S, Denmark.

Solutions:

Assay buffer: 50 mM succinate, 50 mM HEPES, 50 mM CHES, 150 mM KCl, 2 mMCaC₂, 1 mM MgCl₂, 0.01% Triton X100, pH 6.5

GOD-Perid solution: 65 mM sodium phosphate, pH 7, 0.4 g/l Glucoseoxidase, 0.013 g/l HRP (Horse Radish Peroxidase), 0.65 g/l ABTS(2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid)).

Substrate:

160 mM lactose, 50 mM succinate, 50 mM HEPES, 50 mM CHES, 150 mM KCl, 2mM CaCl₂, 1 mom MgCl₂, pH 6.5.

Standard:

Lactozym (available from Novozymes A/S, Denmark) with a known activityin LAU/g is used as standard, diluted in assay buffer in the range from0.09-0.7 LAU/g.

Samples:

Enzyme samples are diluted appropriately in assay buffer.

Procedure:

1. 375 ul substrate is incubated 5 minutes at 37° C.2. 25 ul enzyme diluted in assay buffer is added.3. The reaction is stopped after 30 minutes by adding 60 ul 1 M NaOH4. 20 ul is transferred to a 96 well microtiter plate and 200 ulGOD-Perid solution is added.After 30 minutes at room temperature, the absorbance is measured at 420nm.

Example 2

100 ml 9% skimmed milk solution having approximately 5% lactose was madeby mixing 9 g skimmed milk powder (Kerry) in 91 ml ionic water. 10 ml ofthe solution was transferred to a test tube containing a magneticstirring bar and placed in a water bath at 37° C. After 15 min enzymewas added. Enzymes tested were Lactozym, a commercially availablelactase from Novozymes A/S, Denmark, having an activity of 3060 LAU/g,and an experimental lactase from Bifidobacterium bifidum having theencoded sequence shown in SEQ ID NO: 2 and an activity of 295 LAU/g.Amino acids 1 to 27 of SEQ ID NO: 2 is a signal sequence which ispresumably cleaved off and amino acids 1332 to 1341 is a tag used forpurification of the experimental enzyme.

Dosages were 5640 LAU/l milk of Lactozym and 2700 LAU/l milk of theBifidobacterium lactase. Milk samples were taken at regular intervals uptill 4 hrs. and the enzyme inactivated by heating to 99° C. for 10 minin a thermomixer. Samples were diluted appropriately and filteredthrough a 0.20 um filter.

Lactose hydrolysis was measured using a Dionex BioLC equipped with aDionex PA1 column and a Pulsed Amperiometrisk Detektor (PAD). Peaks wereidentified and quantified by comparing with known standards of lactose,glucose and galactose. Results are given below.

TABLE 1 Lactose, glucose and galactose in reconstituted skimmed milkafter treatment with Lactozym or Bifidobacterium lactase. LactozymGalac- Bifidobacterium lactase Time Lactose Glucose tose Lactose GlucoseGalactose min mM mM mM mM mM mM 5 152 6 5 156 3 3 30 64 92 76 91 71 7060 35 118 99 45 117 114 120 19 131 111 8 144 142 180 15 141 119 1 155153 240 14 150 128 1 162 160

When tested in milk with 5% lactose, no transferase activity is observedwhen using the Bifidobacterium lactase. Glucose and galactose productionare equal and total monosaccharide production match that expected fromthe lactose hydrolyzed. For comparison, Lactozym produces less galactosethan glucose clearly showing that galactooligosaccharides have beenproduced. Also, final lactose levels are significantly lower when usingthe Bifidobacterium lactase illustrating the lower Km value of thisenzyme.

Example 3

100 ml 15 or 30% (w/w) whey permeate containing primarily lactose andions was made by mixing 15 or 30 g spray-dried whey permeate powder(Variolac, Arla) in 85 or 70 ml ionic water respectively. The solutionwas poured in a flask containing a magnetic stirring bar and placed in awater bath at 37° C. After 15 min, enzyme was added. Enzymes tested wereLactozym, a commercially available lactase from Novozymes A/S, Denmark,having an activity of 3060 LAU/g, and an experimental lactase fromBifidobacterium bifidum having the encoded sequence shown in SEQ ID NO:2 and an activity of 295 LAU/g.

Dosages were 4225 LAU/l milk of Lactozym and 2025 LAU/l milk of theBifidobacterium lactase. Milk samples were taken at regular intervals uptill 5.5 hrs. and the enzyme inactivated by heating to 99° C. for 10 minin a thermomixer. Samples were diluted appropriately and filteredthrough a 0.20 um filter.

Lactose hydrolysis was measured using a Dionex BioLC equipped with aDionex PA1 column and a Pulsed Amperiometrisk Detektor (PAD). Peaks wereidentified and quantified by comparing with known standards of lactose,glucose and galactose.

Results are given below.

TABLE 2 Lactose, glucose and galactose in 15% DS whey permeate aftertreatment with Lactozym or Bifidobacterium lactase. LactozymBifidobacterium lactase Time Lactose Glucose Galactose Lactose GlucoseGalactose min mM mM mM mM mM mM 0 499 1 2 499 1 2 30 312 135 106 410 6163 60 211 224 155 349 119 122 120 110 295 221 220 199 202 180 66 324 249149 281 290 240 50 346 279 84 336 348 330 37 372 312 31 350 368

TABLE 3 Lactose, glucose and galactose in 30% DS whey permeate aftertreatment with Lactozym or Bifidobacterium lactase. LactozymBifidobacterium lactase Time Lactose Glucose Galactose Lactose GlucoseGalactose min mM mM mM mM mM mM 0 848 1 4 848 1 4 30 824 109 75 819 4345 60 615 253 150 788 86 83 120 420 370 242 651 159 158 180 291 459 300625 232 230 240 246 559 373 501 283 273 330 154 544 367 391 333 324 144054 649 545 20 727 739

Also when tested at higher lactose concentrations as in 15% or 30% wheypermeate no or very little galactooligosaccharides are produced. Again,the produced galactose and glucose levels are equal and match the amountof lactose hydrolyzed. For comparison, Lactozym produces less galactosethan glucose, clearly showing that galactooligosaccharides have beenproduced.

Example 4

pH Profile (at 37° C.) and Temperature Profile (at pH 6.5) ofExperimental Lactase from Bifidobacterium bifidum Using Lactose asSubstrate.

Principle:

Lactase hydrolyzes lactose and glucose+galactose is formed. Glucose ismeasured after a modified version of the common glucoseoxidase/peroxidase assay (Werner et al., 1970, Z. Analyt. Chem. 252:224.)

pH Profile Substrate:

167 mM lactose, 50 mM succinate, 50 mM HEPES, 50 mM CHES, 150 mM KCl, 2mM CaCl₂, 1 mM MgCl₂ and pH adjusted to pH 3, 4, 5, 6, 7, 8, 9 and 10with NaOH.

Enzyme Sample:

Experimental lactase from Bifidobacterium bifidum having the encodedsequence shown in SEQ ID NO: 2 was diluted appropriately in 150 mM KCl,2 mM CaCl₂, 1 mM MgCl₂, 0.01% Triton X100.

Procedure:

-   -   10 ul enzyme sample diluted in enzyme dilution buffer was added        to PCR tubes at room temperature.    -   90 ul substrate was added at room temp. and quickly placed in a        Peltier Thermal Cycler (PCT-200, MJ research) at 37° C. and        incubated for 30 min and then placed on ice.    -   The reaction was stopped by adding 100 ul 0.25 M NaOH.    -   20 ul was transferred to a 96 well microtitre plate and 230 ul        65 mM sodium phosphate, pH 7, 0.4 g/l Glucose oxidase, 0.013 g/l        HRP, 0.65 g/l ABTS solution was added. After 30 minutes at room        temperature, the absorbance was measured at 420 nm.

TABLE 4 B. bifidum lactase pH relative activity (% of activity at pH6) 30 4 4 5 75 6 100 7 85 8 39 9 10 10 4

Temperature Profile Substrate:

167 mM lactose, 50 mM succinate, 50 mM HEPES, 50 mM CHES, 150 mM KCl, 2mM CaCl₂, 1 mM MgCl₂ and pH adjusted to pH 6.5 with NaOH.

Enzyme Sample:

Experimental lactase from Bifidobacterium bifidum having the encodedsequence shown in SEQ ID NO: 2 was diluted appropriately in 50 mMsuccinate, 50 mM HEPES, 50 mM CHES, 150 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂,0.01% Triton X100 and pH adjusted to pH 6.5.

Procedure:

-   -   10 ul enzyme sample diluted in enzyme dilution buffer was added        to PCR tubes at room temp.    -   90 ul preheated (in a Peltier Thermal Cycler 30-70° C.)        substrate was added and incubation was performed with a temp.        gradient from 30-70° C. for 30 min. and then placed on ice.    -   The reaction was stopped by adding 100 ul 0.25 M NaOH.    -   20 ul was transferred to a 96 well microtitre plate and 230 ul        65 mM sodium phosphate, pH 7, 0.4 g/l Glucose oxidase, 0.013 g/l        HRP, 0.65 g/l ABTS solution was added. After 30 minutes at room        temperature, the absorbance was measured at 420 nm.

TABLE 5 Temp. B. bifidum lactase ° C. relative activity (% of activityat 38.1° C.) 20 54 21 63 22 64 24 68 26 73 29 81 31 88 34 94 36 96 38100 43 96 48 91 52 83 57 76 62 58 66 32 69 20 70 17

Example 5 Determination of Km for Lactase Enzymes at 5° C. Principle:

Lactase hydrolyzes lactose and glucose+galactose is formed. Glucose ismeasured after a modified version of the common glucoseoxidase/peroxidase assay (Werner et al., 1970, Z. Analyt. Chem. 252:224.)

Substrate:

Different lactose concentrations ranging from Km/5 to 10*Km, 50 mMsuccinate, 50 mM HEPES, 50 mM CHES, 150 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂and pH adjusted to pH 6.5 with NaOH.

Enzyme Sample:

Experimental lactase from Bifidobacterium bifidum having the encodedsequence shown in SEQ ID NO: 2 was diluted appropriately in 50 mMsuccinate, 50 mM HEPES, 50 mM CHES, 150 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂,0.01% Triton X100, pH adjusted to pH 6.5 with NaOH.

12 g/l Lactozym (commercially available lactase from Novozymes A/S,Denmark) was diluted 6000 times in the same buffer.

Procedure:

-   -   10 ul enzyme sample (5° C.) was added to a 96 well microtitre        plate on ice.    -   90 ul substrate (5° C.) was added and incubated for 2 hours at        5° C.    -   The reaction was stopped by adding 100 ul 0.25 M NaOH.    -   20 ul was transferred to a 96 well microtitre plate and 230 ul        65 mM sodium phosphate, pH 7, 0.4 g/l Glucose oxidase, 0.013 g/l        HRP, 0.65 g/l ABTS solution was added. After 30 minutes at room        temperature, the absorbance was measured at 420 nm.

Km Determination:

Computerized nonlinear least-squares fitting and the Michaelis-Mentenequation:

v=(V max*S)/(Km+S)

was used. Km for the Bifidobacterium lactase and Lactozym weredetermined to be 8 mM and 30 mM, respectively.

In a similar test performed at 37° C., Km for the Bifidobacteriumlactase and Lactozym were determined to be 13 mM and 30 mM,respectively.

Example 6 Yoghurt Trials

Commercial homogenized milk with 1.5% fat was pasteurized at 90° C. for20 min. 200 ml of the milk was transferred into baby bottles andtempered to 43° C. The milk was inoculated with a frozen probioticyoghurt culture from Chr. Hansen, Denmark, (F-DVS ABY-3) using aninoculation level of 0.02%. At the same time, enzyme was added to themilk. Enzyme products tested were Ha-lactase, a commercially availablelactase from Chr. Hansen, Denmark, having an activity of 8021 LAU/g andan experimental lactase from Bifidobacterium bifidum having the encodedsequence shown in SEQ ID NO: 2 and an activity of 295 LAU/g.

Dosages were 1500, 3000 and 3750 LAU/L milk of Ha-lactase and 710, 1420,and 1780 LAU/L of the Bifidobacterium lactase. The milk samples werefermented at 43° C. until pH reached 4.55 within approximately fivehours. The yoghurts were then stirred, cooled to 25° C. and placed at 8°C. for storage. Samples were collected 2 hours after addition of cultureand enzyme, at end pH (pH 4.55) and after 20-24 hours (Day 1) of storageat 8° C. The biological activity was stopped by addition of sulphuricacid. Proteins were precipitated adding perchloric acid and MQWcontaining standards were then added.

Lactose hydrolysis was measured using a Dionex ICS-3000 system equippedwith a Carbopac20 connected with an electrochemical detector (ED). Peakswere identified and quantified by comparing with known standards oflactose, glucose and galactose. Results for lactose hydrolysis are givenbelow.

TABLE 6 Lactose in yoghurt treated with different dosages of Ha-lactaseor Bifidobacterium lactase. A reference sample with no addition ofenzyme was also tested Lactose (mg/g) Refer- ence HA-lactaseBifidobacterium lactase No 1500 3000 3750 710 1420 1780 Time lactaseLAU/L LAU/L LAU/L LAU/L LAU/L LAU/L Initial 56.0 2 h 48.4 13.3 3.4 2.629.6 10.2 5.7 End 39.0 10.9 2.5 1.9 8.7 0.6 0.6 pH Day 39.3 10.5 2.4 1.83.4 0.5 0.5 1

The level of lactose in the yoghurt samples show that Ha-lactase hashighest activity in the beginning of the fermentation, during the firsttwo hours of fermentation. After two hours Ha-lactase is clearlyinactivated, due to the lowering of pH. The Bifidobacterium lactase, onthe other hand, stays active during the whole fermentation and also tosome extent during cold storage. At the lowest tested dosage of 710LAU/l, the lactose level is significantly reduced during cold storagewhen using the Bifidobacterium lactase.

Example 7 Yoghurt Trials

Commercial homogenized milk with 1.5% fat was pasteurized at 90° C. for20 min. 200 ml of the milk was transferred into baby bottles andtempered to 43° C. The milk was inoculated with a frozen probioticyoghurt culture from Chr. Hansen, Denmark, (F-DVS ABY-3) using aninoculation level if 0.02%. At the same time enzyme was added to themilk. Enzyme products tested were Ha-lactase, a commercially availablelactase from Chr. Hansen, Denmark, having an activity of 8021 LAU/g andan experimental lactase from Bifidobacterium bifidum having the encodedsequence shown in SEQ ID NO: 2 and an activity of 295 LAU/g.

Dosage was 1500 LAU/L milk of Ha-lactase and 710, 530 and 360 LAU/L ofthe Bifidobacterium lactase. The milk samples were fermented at 43° C.until pH reached 4.55 within approximately five hours. The yoghurts werethen stirred, cooled to 25° C. and placed at 8° C. for storage. Sampleswere collected 2 hours after addition of culture and enzyme, at end pH(pH4.55) and after 1, 2, 3 and 7 days of storage at 8° C. The biologicalactivity was stopped by addition of sulphuric acid. Proteins wereprecipitated adding perchloric acid and MQW containing standards wasthen added.

Lactose hydrolysis was measured using a Dionex ICS-3000 system equippedwith a Carbopac20 connected with an electrochemical detector (ED). Peakswere identified and quantified by comparing with known standards oflactose, glucose and galactose. Results for lactose hydrolysis are givenbelow.

TABLE 7 Lactose (mg/g) Ha-lactase Bifidobacterium lactase 1500 710 530360 Time No lactase LAU/L LAU/L LAU/L LAU/L Initial 47.9 2 h 45.8 4.721.2 24.2 29.6 End pH 35.5 2.3 0.8 3.8 9.6 Day 1 33.7 3.1 0.2 0.8 4.7Day 2 2.7 0.5 0.7 2.9 Day 3 2.6 0.3 0.2 1.5 Day 7 2.6 0.1 0.2 0.3

As described in the previous example, the activity period of the twoenzymes tested differs. Ha-lactase shows high activity at the start offermentation whereas the Bifidobacterium lactase stays active during thewhole fermentation time and also during cold storage. Hence, after twodays of storage the lactose level is similar or lower in samples withthe Bifidobacterium lactase compared to the Ha-lactase.

Similar degrees of lactose hydrolysis are obtained day 2 in the yoghurtssamples with 1500 LAU/L Ha-lactase and yoghurt samples with 360 LAU/LBifidobacterium lactase.

Example 8 Milk Trials

Commercial homogenized milk with 1.5% fat was transferred to tubes (10ml) and heated in water baths to 40° C., 50° C. and 55° C.,respectively. Enzyme was then added to the milk samples. Enzyme productstested were Ha-lactase, a commercially available lactase from Chr.Hansen, Denmark, having an activity of 8040 LAU/g and an experimentallactase from Bifidobacterium bifidum having the encoded sequence shownin SEQ ID No. 2 and an activity of 295 LAU/g.

Dosage was 1500 LAU/L milk of Ha-lactase and 710 LAU/L of theBifidobacterium lactase. Samples were collected 2 hours and 4 hoursafter addition of the enzyme. The biological activity was stopped byaddition of sulphuric acid. Proteins were precipitated adding perchloricacid and MQW containing standards was then added.

Lactose hydrolysis was measured using a Dionex ICS-3000 system equippedwith a Carbopac20 connected with an electrochemical detector (ED). Peakswere identified and quantified by comparing with known standards oflactose, glucose and galactose. Results for lactose hydrolysis are givenbelow.

TABLE 8 Lactose (mg/g) Refer- ence HA-lactase - Bifidobacteriumlactase - No 1500 LAU/L 710 LAU/L Time lactase 40° C. 50° C. 55° C. 40°C. 50° C. 55° C. 2 h 46.5 24.0 34.9 40.6 29.3 21.0 31.7 4 h 46.5 19.837.3 39.6 12.5 11.2 25.6

At the highest temperatures, 50° C. and 55° C., the Bifidobacteriumlactase shows significantly higher activity compared to the Ha-lactase.Furthermore, the Bifidobacterium lactase stays active during the 4 hourreaction time, whereas no or only very low activity is observed for theHa-lactase.

Example 9 Milk Trials—High Temperature

Commercial homogenized milk with 1.5% fat was transferred to tubes (10ml) and tempered to 63° C. Enzyme was added to the milk samples. Enzymeproducts tested were Ha-lactase 5200, a commercially available lactasefrom Chr. Hansen (Denmark) having an activity of 8040 LAU/g andLactoles, a commercial Bacillus lactase from Daiwa Kasei (Japan) havingan activity of approximately 1500 LAU/g.

Applied dosages were 1500 LAU/L milk of Ha-lactase and Lactoles,respectively. At 63° C. samples were collected 15 minutes, 30 minutes, 2hours and 4 hours after addition of the enzyme. The enzymatic activityin the samples was stopped by addition of sulphuric acid and proteinsprecipitated by addition of perchloric acid before HPLC analysis.

Lactose hydrolysis was measured using a Dionex ICS-3000 system equippedwith a Carbopac20 connected with an electrochemical detector (ED). Peakswere identified and quantified by comparing with known standards oflactose, glucose and galactose. Results for lactose hydrolysis are givenbelow.

TABLE 9 Lactose (mg/g) Reference- HA-lactase 5200 - Lactoles - Time Nolactase 1500 LAU/L 1500 LAU/L 15 min. 48.9 44.6 22.7 30 min. 48.9 44.922.1  2 h 48.9 44.0 6.7  4 h 48.9 43.2 3.2

At 63° C., Ha-lactase 5200 is inactivated as no hydrolysis takes placeduring 4 hours of reaction. On the other hand, Lactoles shows highactivity at this temperature during the whole reaction time. After 4hours, a degree of hydrolysis of 93.4% is obtained.

Example 10

100 ml 9% skimmed milk solution having approximately 5% lactose was madeby mixing 9 g skimmed milk powder (Kerry) in 91 ml ionic water. 10 ml ofthe solution was transferred to a test tube containing a magneticstirring bar and placed in a water bath at 5° C. After 15 min enzyme wasadded. Enzymes tested were Lactozym, a commercially available lactasefrom Novozymes A/S, Denmark, having an activity of 3060 LAU/g, and anexperimental lactase from Bifidobacterium bifidum having the encodedsequence shown in SEQ ID NO: 2 and an activity of 295 LAU/g. Amino acids1 to 27 of SEQ ID NO: 2 is a signal sequence which is presumably cleavedoff and amino acids 1332 to 1341 is a tag used for purification of theexperimental enzyme.

Dosages were 3000 LAU/I milk of Lactozym and 1420 LAU/I milk of theBifidobacterium lactase. Milk samples were taken at regular intervals uptill 48 hrs. and the enzyme inactivated by heating to 99° C. for 10 minin a thermomixer. Samples were diluted appropriately and filteredthrough a 0.20 um filter.

Lactose hydrolysis was measured using a Dionex BioLC equipped with aDionex PA1 column and a Pulsed Amperiometrisk Detektor (PAD). Peaks wereidentified and quantified by comparing with known standards of lactose,glucose and galactose. Results are given below.

TABLE 10 Lactose, glucose and galactose in reconstituted skimmed milkafter treatment with Lactozym or Bifidobacterium lactase at 5° C.Lactozym Bifidobacterium lactase Time Lactose Glucose Galactose LactoseGlucose Galactose min mM mM mM mM mM mM 125 — — — 110 28 31 240 87 64 60— — — 360 — — — 70 65 67 460 62 104 91 55 74 76 1410 18 152 134 13 149148 1620 13 137 125 7 139 140 1865 10 — — 5 167 167 2870 6 141 132 0.7139 140

When tested in milk with 5% lactose at 5° C. again no transferaseactivity is observed when using the Bifidobacterium lactase. Glucose andgalactose production are equal and total monosaccharide production matchthat expected from the lactose hydrolyzed. For comparison, Lactozymproduces less galactose than glucose clearly showing thatgalactooligosaccharides have been produced. Also, final lactose levelsare significantly lower when using the Bifidobacterium lactase furtherillustrating the lower Km value of this enzyme.

1. A method for producing a dairy product comprising a) providing amilk-based substrate comprising lactose; and b) treating said substratewith an enzyme having lactase activity and having an amino acid sequencewhich is at least 70% identical to amino acids 28-1331 of SEQ ID NO: 2.2. A method for producing a dairy product comprising a) providing amilk-based substrate comprising lactose; and b) treating said substratewith an enzyme having lactase activity and having an amino acid sequencewhich is at least 70% identical to amino acids 28-1931 of SEQ ID NO: 1or a fragment thereof.
 3. A method for producing a dairy productcomprising a) providing a milk-based substrate comprising lactose; andb) treating said substrate with an enzyme having lactase activity andhaving an amino acid sequence which is at least 70% identical to any ofSEQ ID NO's: 3-4 or a fragment of any of these. 4-24. (canceled)